DNA-mediated charge transport (DNA CT) provides an avenue for long-range, rapid signaling between redox-active moieties coupled into duplex DNA. Several DNA-processing enzymes have moreover been shown to contain [4Fe4S] clusters, common redox cofactors. Eukaryotic DNA primase, the heterodimeric enzyme responsible for initiating DNA replication, contains a [4Fe4S] cluster in the C-terminal domain of the large subunit (p58C). Primase synthesizes a short RNA primer on a single-stranded DNA template and subsequently hands this template off to DNA polymerase α, another [4Fe4S] protein, through a mechanism which is unclear. Here we show electrochemical evidence that the [4Fe4S] cluster in the p58C domain of human DNA primase performs redox chemistry on DNA, cycling reversibly between a tightly DNA-bound, oxidized [4Fe4S]3+ state, and a loosely associated, reduced [4Fe4S]2+ state. We demonstrate through structural, biochemical, and electrochemical comparisons of wild type and mutant p58C that the redox switch is mediated by a pathway of tyrosine residues between the cluster and bound DNA. Charge transfer pathway mutations in full-length primase, additionally, abrogate initiation of primer synthesis on single-stranded DNA but do not affect nucleotide polymerization. We further compare primer elongation on a well-matched and mismatched DNA template, showing that a single-base mismatch in the nascent primer inhibits primase termination. Thus primer termination appears to be gated by mismatch-sensitive DNA charge transport. Based on our experimental evidence, we propose a model in which electron transfer between [4Fe4S] clusters, gated by DNA-mediated charge transport, regulates DNA binding and substrate handoff between primase and polymerase α to begin replication.

Functional role for the [4Fe4S] cluster in human DNA primase as a redox switch using DNA charge transport

254th National Meeting (2017)

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